Abstract:

The present invention provides an improved coating for surfaces of medical
implants. The coating comprises at least one interfacial biomaterial
(IFBM) which is comprised of at least one binding module that binds to
the surface of an implant or implant-related material ("implant module")
and at least one binding module that selectively binds to a target
analyte or that is designed to have a desired effect ("analyte module").
The modules are connected by a linker. In some embodiments, the IFBM
coating acts to promote the recognition and attachment of target analytes
to surface of the device. The IFBM coating improves the performance of
implanted medical devices, for example, by promoting osteointegration of
the implant.

Claims:

1.-19. (canceled)

20. A binding module comprising a polypeptide selected from the group
consisting of SEQ ID NOs: 25-26 and 77-81, or a conservatively
substituted variant thereof, wherein the binding module binds to a bone
morphogenetic protein.

21. A binding module comprising a polypeptide having at least 70% sequence
identity to any one of SEQ ID NOs: 25-28 or 77-81, wherein the binding
module binds to a bone morphogenetic protein.

23. An isolated nucleic acid comprising a nucleotide sequence encoding an
amino acid sequence selected from the group consisting of SEQ ID NOs:
25-26 and 77-81, or a conservatively substituted variant thereof.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a divisional application of U.S. Non-provisional
application Ser. No. 12/488,183, filed on Jun. 19, 2009, which is a
divisional application of U.S. Non-provisional application Ser. No.
11/152,974, filed on Jun. 15, 2005, now U.S. Pat. No. 7,572,766, which
claims priority to U.S. Provisional Application No. 60/580,019, filed
Jun. 16, 2004; U.S. Provisional Application No. 60/651,338, filed Feb. 9,
2005; and U.S. Provisional Application No. 60/651,747, filed Feb. 10,
2005; each of which is hereby incorporated in its entirety by reference
herein.

FIELD OF THE INVENTION

[0002]The present invention provides materials and methods for coating
surfaces of medical devices with interfacial biomaterials that promote
the specific recognition and attachment of the target analyte to the
surface of the device.

BACKGROUND OF THE INVENTION

[0003]Orthopedic implants are used for a variety of joint replacements and
to promote bone repair in humans and animals. According to medical
industry analysts, there are now over 800,000 hip and knee joint
replacements performed in human patients each year in the U.S. In
addition, hundreds of thousands of human patients undergo surgical
procedures in which orthopedic implants are used, for example, to treat
various types of bone fractures or to relieve severe back pain.

[0004]With all of these procedures, there is a need for controlled,
directed, rapid healing. Individuals undergoing joint replacement often
experience uncomplicated healing and restoration of function.
Unfortunately, there is a high rate of complications, including "late
failures." The revision surgery rate for human total joint replacement
varies between 10 to 20% (Malchau et al. (2002) "Prognosis of total hip
replacement: Update of results and risk-ratio analysis for revision and
re-revision from the Swedish National Hip Arthroplasty Registry,
1979-2000," scientific exhibition at the 69th Annual Meeting of the
American Academy of Orthopaedic Surgeons, Dallas, Tex., Feb. 13-17, 2002;
Fitzpatrick et al. (1998) Health Technol. Assess. 2:1-64; Mahomed et al.
(2003) J. Bone Joint Surg. Am. 85-A:27-32)). The majority of these
revision surgeries are made necessary by failure at the implant-bone
interface.

[0005]Orthopedic implants are made of materials which are relatively inert
("alloplastic" materials), typically metallic, ceramic, or plastic
materials. Previous approaches to improve the outcomes of orthopedic
implant surgeries have mainly focused on physical changes to the implant
surface that result in increased bone formation. These approaches include
using implants with porous metallic surfaces to promote bone ingrowth and
spraying implants with hydroxyapatite plasma. Approaches using dental
implants have also included the use of topographically-enhanced titanium
surfaces in which surface roughness is imparted by a method such as grit
blasting, acid etching, or oxidation. While these techniques have
improved the outcomes of orthopedic implant surgeries, there is still
considerable room for further improvement.

[0006]Tissue response to an alloplastic material is known to be influenced
by cell adhesion to the material's surface, and much research has been
directed to improving cell adhesion to alloplastic materials. Cell
adhesion between cells in vivo is known to be controlled primarily by the
binding of short, exposed protein domains in the extracellular matrix to
cell surface receptors (LeBaron & Athanasiou (2000) Tissue Eng. 6:
85-103; Yamada (1997) Matrix Biol. 16: 137-141). Notably, a class of
receptors known as integrins has been implicated in cell adhesion to
implant surfaces. Integrins and their target ligands have been shown to
stimulate osteoblast adhesion and proliferation as well as bone formation
(see, e.g., Kantlehner et al. (2000) ChemBioChem 1: 107-114; Sarmento et
al. (2004) J. Biomed. Mater. Res. 69A: 351-358; Hayashibara et al. (2004)
J. Bone Mineral Res. 19: 455-462. Integrins may be useful in targeting
cell adhesion to implants and in this manner may improve integration of
implants into adjacent bone.

[0007]Other research has shown that the local expression of growth factors
and cytokines can enhance tissue reactions at alloplastic implant
surfaces. For example, Cole et. al. ((1997) Clin. Orthop. 345: 219-228)
have shown that growth factors can promote the integration of an implant
into adjacent bone ("osteointegration") as well as increase the rate of
bone formation next to the implant surface. See also U.S. Pat. No.
5,344,654. Growth factors that stimulate new bone production
("osteoinductive proteins") include, but are not limited to,
platelet-derived growth factor (PDGF), insulin-like growth factors 1 and
2 (IGF-1 and IGF-2), vascular endothelial growth factor (VEGF),
fibroblast growth factor (FGF), transforming growth factor (TGF-β),
bone morphogenic proteins (BMP), and associated family members.

[0008]The most effective osteoinductive proteins are the bone
morphogenetic proteins (BMPs). The BMPs are members of the TGF-β
superfamily that share a set of conserved cysteine residues and a high
level of sequence identity overall. Over 15 different BMPs have been
identified, and most BMPs stimulate the cascade of events that lead to
new bone formation (see U.S. Pat. Nos. 5,013,649; 5,635,373; 5,652,118;
and 5,714,589; also reviewed by Reddi and Cunningham (1993) J. Bone
Miner. Res. 8 Supp. 2: S499-S502; Issack and DiCesare (2003) Am. J.
Orthop. 32: 429-436; and Sykaras & Opperman (2003) J. Oral Sci. 45:
57-73). This cascade of events that leads to new bone formation includes
the migration of mesenchymal stem cells, the deposition of
osteoconductive matrix, the proliferation of osteoprogenitor cells, and
the differentiation of progenitor cells into bone-producing cells. Much
research has been directed to the use of BMPs on or near implants in
order to promote osteointegration of the implants (see, e.g.: Friedlander
et al. (2001) J. Bone Joint Surg. Am. 83-A Suppl. 1 (Pt. 2): S151-58;
Einhorn (2003) J. Bone Joint Surg. Am. 85-A Supp1.3: 82-88; Burkus et al.
(2002) J. Spinal Disord. Tech. 15(5): 337-49). However, one of the
critical issues that remains unresolved is the method of grafting or
immobilizing an active BMP or other active biomolecule onto the surface
of an implant.

[0009]It has been shown that the presentation of BMPs is critical for
producing desired bone formation next to an implant device. Approaches to
improving implants have been modeled in view of the natural process of
bone formation. In human bone, collagen serves both as a scaffold for
bone formation and as a natural carrier for BMPs. Demineralized bone has
been used successfully as a bone graft material; the main components of
demineralized bone are collagen and BMPs (see U.S. Pat. No. 5,236,456).
Many matrix systems have been developed that are designed to encourage
bone formation by steadily releasing growth factors and other bioactive
molecules as the matrix degrades. The efficiency of BMP release from
polymer matrixes depends on matrix characteristics such as the affinity
of BMP for the matrix, resorbtion rate, density, and pore size. Materials
used in such matrix systems include organic polymers which readily
hydrolyze in the body into inert monomers. Such organic polymers include
polylactides, polyglycolides, polyanhydrides, and polyorthoesters (see
U.S. Pat. Nos. 4,563,489; 5,629,009; and 4,526,909). Other materials
described as being useful in BMP-containing matrices include polylactic
and polyglycolic acid copolymers, alginate, poly(ethylene glycol),
polyoxyethylene oxide, carboxyvinyl polymer, and poly (vinyl alcohol)
(see U.S. Pat. No. 5,597,897). Natural matrix proteins have also been
used to deliver BMPs to bone areas; these natural proteins include
collagen, glycosaminoglycans, and hyaluronic acid, which are
enzymatically digested in the body (see U.S. Pat. Nos. 4,394,320;
4,472,840; 5,366,509; 5,606,019; 5,645,591; and 5,683,459).

[0010]Even with the use of a polymer matrix to retain BMP at the site of
repair, it has been found that supraphysiological levels of BMP are
required in order to promote healing due to the rapid diffusion of growth
factors out of the matrix. For example, with a collagen sponge delivery
system, only 50% of the BMP added to the sponge is retained after two
days (Geiger et al. (2003) Adv. Drug Del. Rev. 55: 1613-1629). The high
initial dose of BMPs required to maintain physiological levels of BMP for
the necessary period of time makes BMP treatment more expensive and may
lead to detrimental side effects such as ectopic bone formation or
allergic reactions, or the formation of neutralizing antibodies.

[0012]Thus, there remains a need for the development of cost-effective
methods for grafting active biomolecules to the surface of materials used
as implants or in conjunction with implants in order to promote
post-surgical healing and, where desirable, integration of the implant
into surrounding tissues, such as, for example, adjacent bone.

SUMMARY OF THE INVENTION

[0013]The present invention provides an improved coating for surfaces of
medical implants. The coating comprises at least one interfacial
biomaterial (IFBM) which is comprised of at least one binding module that
binds to the surface of an implant or implant-related material ("implant
module") and at least one binding module that binds to a target analyte
or that is designed to have a desired effect ("analyte module"). The
modules are connected by a linker. In some embodiments, the IFBM coating
acts to promote the recognition and attachment of target analytes to
surface of the device. The IFBM coating improves the performance of
implanted medical devices by promoting osteointegration of the implant,
accelerating healing, and/or reducing inflammation at the site of the
implant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows a comparison of the binding of phage that display
representative titanium-binding peptides to titanium beads (see Example
1). Signal of assay for binding to titanium beads (vertical axis) is
shown for various phage (horizontal axis).

[0015]FIG. 2 shows a comparison of the binding of peptides with a
C-terminal biotin residue to titanium (see Example 1). Absorbance
(vertical axis) is shown as a function of peptide concentration (μM,
on the horizontal axis).

[0016]FIG. 3 shows a comparison of binding to titanium of two peptides
(see Example 2). A405 nm signal (vertical axis) is shown as a function of
peptide concentration (μM, on the horizontal axis). The lines shown on
the graph from top to bottom join data points for peptides AFF6007 and
AFF6010, respectively.

[0017]FIG. 4 shows a comparison of binding of various peptides to BMP-2
(see Example 3). Signal (rate AP) is shown for various peptides
(identified on the horizontal axis).

[0018]FIG. 5 shows the effect of BMP on the binding of IFBMs to a collagen
sponge (see Example 4). Signal (vertical axis) is shown as a function of
BMP concentration in nM (horizontal axis).

[0019]FIGS. 6A, 6B, 6C, and 6D show the results of an experiment described
in Example 4 which demonstrates that the binding of BMP to collagen via
an IFBM is dependent on both the amount of BMP put into the sponge and
also on the amount of IFBM present. Absorbance (vertical axis) is shown
as a function of BMP concentration (horizontal axis).

[0020]FIG. 7 shows the results of an analysis of all the peptide sequences
from Tables 3 and 4 that bind BMP-2 and contain Motif 1 (see Example 3).
The figure shows for each analyzed position the number of times each
amino acid was found in that position in the peptide sequences analyzed;
for example, "G2" in position 1 means that Glycine was found two times in
that position.

[0021]FIG. 8 shows the oligonucleotide cassette which was designed to
express a peptide (SEQ ID NO: 74) containing the core binding Motif 1a in
the context of a peptide sequence which also contained consensus residues
identified for other positions in the sequence (see Example 3). The
nucleotide sequences shown in the figure are also set forth in SEQ ID NO:
75 and SEQ ID NO: 76.

[0022]FIG. 9 shows results from a conventional ELISA performed to evaluate
the relative affinity of BMP binding peptides (see Example 3). The signal
from the ELISA (A405 nm reading) is presented on the vertical axis as a
function of microliters of phage on the horizontal axis. At the data
points corresponding to 0.10 microliters of phage, the lines shown on the
graph from top to bottom join data points for: APO2-61, APO2-40, APO2-41,
APO2-26, APO2-35, APO2-59, APO2-44, mAEK, and the no-phage control,
respectively.

[0023]FIG. 10 shows the results of an analysis of all the peptide
sequences from Tables 3 and 5 that bind BMP-2 and contain Motif 2. The
figure shows for each analyzed position the number of times each amino
acid was found in that position in the peptide sequences analyzed; for
example, "G7" in position 1 means that Glycine was found seven times in
that position. Also shown are a consensus sequence derived from an
alignment of the peptides from Tables 3 and 5 that contain Motif 2 (SEQ
ID NO: 93). This sequence represents the predominant amino acid found at
each position after all the peptides are aligned. Among the sequences
examined, the most conserved amino acids form a core binding motif
designated "Motif 2a" (SEQ ID NO: 94).

[0024]FIG. 11 shows representative results from an alternate assay for
BMP-binding activity in which binding occurs in the solution phase (see
Example 3). Absorbance at 405 nm (vertical axis) is shown as a function
of picomoles of BMP (horizontal axis). These results were used to
calculate the affinity of each BMP-binding peptide for BMP-2 (see Table
6). At the data point corresponding to one picomole of BMP, the lines
shown on the graph from top to bottom join data points for: 2006, 2007,
2008, 2009, 2011, and 2012, respectively.

[0025]FIG. 12 shows results from an assay in which several peptides were
tested for their ability to bind to BMP-2, BMP-4, and BMP-7 (see Example
3). The 2007 and 2011 peptides were originally identified as BMP-2
binding peptides, while the 9001 peptide was originally identified as
binding to an unrelated target.

DETAILED DESCRIPTION OF THE INVENTION

[0026]The present invention provides an improved coating for surfaces of
medical devices to promote the attachment of peptides, proteins, drugs,
or cells to the device. The coating is an interfacial biomaterial (IFBM)
that comprises multiple binding modules that are linked. The IFBM
comprises at least one binding module which binds to the surface of the
implant ("implant module") and at least one binding module that binds to
a target analyte or has a desired effect ("analyte module"). Exemplary
binding modules comprise the peptide sequences provided, for example, in
the sequence listing (SEQ ID NOs: 1-74 and 77-558). The modules are
connected by a linker. In some embodiments, the binding of the binding
module of an IFBM to the surface of an implant is non-covalent.
Similarly, in some embodiments, the binding of an analyte module to a
target analyte is non-covalent. According to one embodiment, the implant
module and the analyte module comprise two separate peptide molecules
such that the implant module binds to an implant material and the analyte
module binds specifically to a growth factor or cell. In some
embodiments, the implant module and the analyte module are linked by a
central macromolecule. These binding modules typically bind
non-covalently to the implant material or target analyte, respectively.
In embodiments where the analyte module does not bind to a target analyte
but rather has a desired effect, the analyte module may, for example,
simulate the action of a growth factor by acting to recruit cells to the
location of the implant. The IFBM selection method and structure are
described in U.S. patent application Ser. No. 10/300,694, filed Nov. 20,
2002 and published on Oct. 2, 2003 as publication number 20030185870,
which is herein incorporated by reference.

[0027]By "binds specifically" or "specific binding" is intended that the
implant module or analyte module binds to a selected implant material or
to a selected analyte. In some embodiments, a module that binds
specifically to a particular implant material or analyte binds to that
material or analyte at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 200%, 300%, 400%, 500%, or a higher percentage more than the module
binds to an appropriate control such as, for example, a different
material that is used in implants, a material that is not used in
implants, or a protein typically used for such purposes such as bovine
serum albumin. By "analyte" is intended any substance or moiety that
improves osteointegration of an implant or promotes or accelerates
healing of the surrounding tissues following implant surgery. Suitable
analytes which are binding targets for analyte modules include, but are
not limited to, growth factors such as bone morphogenic proteins (BMPs,
such as, for example, BMP-7 and BMP-2), vascular endothelial growth
factor (VEGF), platelet-derived growth factor (PDGF), transforming growth
factor-β (TGF-β), insulin growth factor-1 (IGF-1), insulin
growth factor-2 (IGF-2), fibroblast growth factor (FGF), nerve growth
factor (NGF), and placental growth factor. Suitable analytes also include
hormones, enzymes, cytokines, and other bioactive substances or moieties
which are useful in obtaining the goals of the invention; that is, to
promote osteointegration of an implant and/or to improve healing of
surrounding tissues following implant surgery. Suitable analytes also
include cells, for example, osteoblasts, chondrocytes, stem cells,
progenitor cells, platelets, and other cells which perform roles in
osteointegration and healing. In some embodiments, analyte modules can
comprise peptide sequences that bind cells or have bioactivity through
binding to cells or receptors such as, for example, the peptide sequences
RGD, YIGSR, and IKVAV, which are known in the art to have particular
biological activities. See, e.g., Hersel et al. (2003) Biomaterials 24:
4385-4415; Grant et al. (1990) Ann. N.Y. Acad. Sci. 588: 61-72; Hosokawa
et al. (1999) Dev. Growth Differ. 41: 207-216. In some embodiments,
analyte modules comprise peptide sequences which bind to and/or mimic the
effect of BMP-2, such as the exemplary sequences set forth in SEQ ID NOs:
11-28, 44-74, or 77-94. An analyte module that binds to cells can
comprise a peptide that comprises a general cell attachment sequence that
binds to many different cell types, or it can comprise a peptide that
binds to a specific cell type such as an osteoblast, a chondrocyte, an
osteoprogenitor cell, or a stem cell.

[0028]The term "implant" generally refers to a structure that is
introduced into a human or animal body to restore a function of a damaged
tissue or to provide a new function. An implant device can be created
using any biocompatible material to which binding agents can specifically
bind as disclosed herein. Representative implants include but are not
limited to: hip endoprostheses, artificial joints, jaw or facial
implants, tendon and ligament replacements, skin replacements, bone
replacements and artificial bone screws, bone graft devices, vascular
prostheses, heart pacemakers, artificial heart valves, breast implants,
penile implants, stents, catheters, shunts, nerve growth guides,
intraocular lenses, wound dressings, and tissue sealants. Implants are
made of a variety of materials that are known in the art and include but
are not limited to: a polymer or a mixture of polymers including, for
example, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic
acid copolymers, polyanhidrides, polyorthoesters, polystyrene,
polycarbonate, nylon, PVC, collagen (including, for example, processed
collagen such as cross-linked collagen), glycosaminoglycans, hyaluronic
acid, alginate, silk, fibrin, cellulose, and rubber; plastics such as
polyethylene (including, for example, high-density polyethylene (HDPE)),
PEEK (polyetheretherketone), and polytetrafluoroethylene; metals such as
titanium, titanium alloy, stainless steel, and cobalt chromium alloy;
metal oxides; non-metal oxides; silicone; bioactive glass; ceramic
material such as, for example, aluminum oxide, zirconium oxide, and
calcium phosphate; other suitable materials such as demineralized bone
matrix; and combinations thereof. The term "polymer" as used herein
refers to any of numerous natural and synthetic compounds of usually high
molecular weight consisting of up to millions of repeated linked units,
each a relatively simple molecule. The term "implant" as used herein
includes implant-related materials that are associated with the implant
and are also introduced into a human or animal body in conjunction with
the implant.

[0029]In one embodiment of the invention, an IFBM creates a binding
interface that mediates the attachment of growth factors to the surface
of an implant. In some embodiments, implants prepared according to the
methods of the invention will have growth factors specifically attached
to the surface of the implant; the rate of diffusion of the growth factor
away from the site of the implant can vary depending on the affinity of
the analyte module for the growth factor in question and thus implants
can be prepared with varying rates of diffusion of growth factors. In
embodiments involving the attachment of growth factors to the surface of
an implant, the growth factor will have a positive effect such as, for
example, accelerating the healing process, reducing the amount of growth
factor required for healing, and minimizing the side effects caused by
using supraphysiological doses of the growth factor. Growth factors of
particular interest either as analyte modules or as factors that bind to
analyte modules include, for example, BMP-2, BMP-7, PDGF, FGF, and
TGFβ.

[0030]Thus, the present invention provides methods for preparing an
implant to be surgically placed into a patient wherein the device is
coated with a layer comprising at least one IFBM. In some embodiments,
the method comprises the steps of: (a) applying an IFBM coating to the
implant, wherein the IFBM comprises an implant module that specifically
binds to the implant and an analyte module that specifically binds a
growth factor; (b) applying the growth factor to the surface of the
implant by dipping, spraying, or brushing a solution containing the
growth factor onto the implant; (c) placing the implant into a subject
using appropriate surgical techniques which will be known to those of
skill in the art.

[0031]Alternatively, a method for coating an implant so that the implanted
device promotes growth factor attachment comprises the steps of: (a)
applying an IFBM coating to the implant, wherein the IFBM comprises an
implant module that specifically binds the implant and an analyte module
that specifically binds growth factor at an implant site; and (b) placing
the implant in a subject at the implant site; whereby growth factor
produced in the host binds to the implant via the IFBM. The enhanced
presence of growth factor at the implant site enhances healing of
adjacent tissue and integration of the implant into the adjacent tissue.

[0032]In one embodiment of the invention, an IFBM mediates cell attachment
to the surface of an implant. By enhancing cell adhesion and tissue
integration, the IFBMs of the invention can accelerate healing and
improve the function of the implanted device. Thus, in accordance with
the present invention, a method for preparing an implant to be surgically
placed into a patient can comprise: (a) applying an IFBM coating to the
implant, wherein the IFBM comprises at least one implant module that
specifically binds the implant and at least one analyte module that
specifically binds to at least one type of cell; and (b) placing the
implant in a subject at the implant site, whereby cells bind to the IFBM
coating on the implant.

[0033]In some embodiments, a method for preparing an implant comprises:
(a) applying an IFBM coating to the implant, wherein the IFBM comprises
at least one implant module that specifically binds the implant and at
least one analyte module that specifically binds at least one type of
cell; and (b) applying cells to the surface of the implant, for example,
by dipping the implant into a solution containing the cells or brushing a
solution containing the cells onto the implant. The implant may then be
placed into a subject (i.e., a human patient or an animal patient). By
"patient" as used herein is intended either a human or an animal patient.

[0034]In another embodiment of the invention, an implant is coated with
more than one type of IFBM in order to provide a coating with multiple
functionalities. For example, an implant coating can comprise a first
IFBM having an analyte module that binds a cell and a second IFBM having
an analyte module that binds a growth factor. A coating comprising these
IFBMs would bind both cells and growth factor to the surface of the
implant. In some embodiments, these IFBMs would be intermingled in the
coating so that the bound growth factor is in close proximity to the
bound cells. In one embodiment, a coating comprises an IFBM that binds to
mesenchymal stem cells and an IFBM that binds to the growth factor BMP-2;
the BMP-2 would trigger the differentiation of the stem cells into
osteoblasts. In other embodiments, an implant coating can comprise a
mixture of at least two different IFBMs which differ in either or both
their implant module and their analyte module. In another embodiment, a
coating comprises a multi-functional IFBM which has two analyte modules,
one of which binds to a cell and one of which binds to a growth factor.

[0035]Binding modules (i.e., implant modules and/or analyte modules) may
be peptides, antibodies or antibody fragments, polynucleotides,
oligonucleotides, complexes comprising any of these, or various molecules
and/or compounds. Binding modules which are peptides may be identified as
described in pending U.S. patent application Ser. No. 10/300,694, filed
Nov. 20, 2002 and published on Oct. 2, 2003 as publication number
20030185870. In some embodiments, binding modules may be identified by
screening phage display libraries for binding to materials including
biocompatible materials (i.e., "biomaterials") such as titanium,
stainless steel, cobalt-chrome alloy, polyurethane, polyethylene or
silicone.

[0036]In some embodiments of the invention, the analyte module is a
bioactive peptide or binds to a bioactive peptide. These bioactive
peptides may be fragments of native proteins that retain the biological
effect of the native protein, as is well-known in the art. For example,
TP508 is a synthetic peptide derived from thrombin which represents amino
acids 183-200 of human thrombin and has been shown to accelerate fracture
healing (see, e.g., Wang et al. (2002) Trans ORS 27: 234). TP508 function
is believed to be mediated by an RGD sequence within the peptide that
binds to integrins present on the cell surface (see, e.g., Tsopanoglou et
al. (2004) Thromb Haemost. 92(4):846-57.) Similarly, P-15 is a 15 amino
acid peptide derived from Type I collagen that represents the
cell-binding domain of collagen (see, e.g., Yang et al. (2004) Tissue
Eng. 10(7-8): 1148-59). P-15 has been shown to enhance new bone formation
(see, e.g., Scarano et al. (2003). Implant Dent. 12(4): 318-24.).
Bioactive peptides can also be fragments of growth factors. For example,
Saito et al. (J Biomed Mater Res A. 2005 72A(1): 77-82) have shown that a
synthetic peptide representing amino acids 73-92 of BMP-2 retains BMP-2
biological activities including binding to a BMP-2 receptor, activating
gene expression and inducing ectopic bone formation.

[0037]Any implant module may be combined with any analyte module to create
an IFBM of the invention so long as the desired activity is provided;
that is, so long as the IFBM specifically binds to a suitable implant and
has a suitable effect conferred by the analyte module, i.e., the ability
to bind to BMP-2. One of skill in the art will appreciate that a variety
of types and numbers of implant modules may be combined with a variety of
types and numbers of analyte modules to create an IFBM of the invention.
Thus, for example, one or more implant modules may be linked with one or
more analyte modules to create an IFBM. One of skill will be able to
select suitable implant module(s) and analyte module(s) depending on the
material of which an implant is made and the desired activity to be
conferred by the analyte module(s).

[0038]The term "antibody" as used herein includes single chain antibodies.
Thus, an antibody useful as a binding module may be a single chain
variable fragment antibody (scFv). A single chain antibody is an antibody
comprising a variable heavy and a variable light chain that are joined
together, either directly or via a peptide linker, to form a continuous
polypeptide. The term "single chain antibody" as used herein encompasses
an immunoglobulin protein or a functional portion thereof, including but
not limited to a monoclonal antibody, a chimeric antibody, a hybrid
antibody, a mutagenized antibody, a humanized antibody, and antibody
fragments that comprise an antigen binding site (e.g., Fab and
Fv antibody fragments).

[0039]Phage display technology is well-known in the art. Using phage
display, a library of diverse peptides can be presented to a target
substrate, and peptides that specifically bind to the substrate can be
selected for use as binding modules. Multiple serial rounds of selection,
called "panning," may be used. As is known in the art, any one of a
variety of libraries and panning methods can be employed to identify a
binding module that is useful in the methods of the invention. For
example, libraries of antibodies or antibody fragments may be used to
identify antibodies or fragments that bind to particular cell populations
or to viruses (see, e.g., U.S. Pat. Nos. 6,174,708; 6,057,098; 5,922,254;
5,840,479; 5,780,225; 5,702,892; and 5,667,988). Panning methods can
include, for example, solution phase screening, solid phase screening, or
cell-based screening. Once a candidate binding module is identified,
directed or random mutagenesis of the sequence may be used to optimize
the binding properties of the binding module. The terms "bacteriophage"
and "phage" are synonymous and are used herein interchangeably.

[0040]A library can comprise a random collection of molecules.
Alternatively, a library can comprise a collection of molecules having a
bias for a particular sequence, structure, or conformation. See, e.g.,
U.S. Pat. Nos. 5,264,563 and 5,824,483. Methods for preparing libraries
containing diverse populations of various types of molecules are known in
the art, and numerous libraries are also commercially available. Methods
for preparing phage libraries can be found, for example, in Kay et al.
(1996) Phage Display of Peptides and Proteins (San Diego, Academic
Press); Barbas (2001) Phage Display: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.)

[0042]A peptide useful as a binding module can be subject to various
changes, substitutions, insertions, and deletions where such changes
provide for certain advantages in its use. Thus, the term "peptide"
encompasses any of a variety of forms of peptide derivatives including,
for example, amides, conjugates with proteins, cyclone peptides,
polymerized peptides, conservatively substituted variants, analogs,
fragments, chemically modified peptides, and peptide mimetics. Any
peptide that has desired binding characteristics can be used in the
practice of the present invention.

[0044]Representative derivatized amino acids include, for example, those
molecules in which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free
carboxyl groups can be derivatized to form salts, methyl and ethyl esters
or other types of esters or hydrazides. Free hydroxyl groups can be
derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen
of histidine can be derivatized to form N-im-benzylhistidine.

[0045]The term "conservatively substituted variant" refers to a peptide
having an amino acid residue sequence substantially identical to a
sequence of a reference peptide in which one or more residues have been
conservatively substituted with a functionally similar residue such that
the "conservatively substituted variant" will bind to the same binding
partner with substantially the same affinity as the parental variant and
will prevent binding of the parental variant. In one embodiment, a
conservatively substituted variant displays a similar binding specificity
when compared to the reference peptide. The phrase "conservatively
substituted variant" also includes peptides wherein a residue is replaced
with a chemically derivatized residue.

[0046]Examples of conservative substitutions include the substitution of
one non-polar (hydrophobic) residue such as isoleucine, valine, leucine
or methionine for another; the substitution of one aromatic residue such
as tryptophan, tyrosine, or phenylalanine for another; the substitution
of one polar (hydrophilic) residue for another such as between arginine
and lysine, between glutamine and asparagine, between glycine, alanine,
threonine and serine; the substitution of one basic residue such as
lysine, arginine or histidine for another; or the substitution of one
acidic residue such as aspartic acid or glutamic acid for another.

[0047]While exemplary peptide sequences for use as binding modules in
IFBMs of the invention are disclosed herein (e.g., in the sequence
listing in SEQ ID NOs: 1-74 and 77-558), one of skill will appreciate
that the binding or other properties conferred by those sequences may be
attributable to only some of the amino acids comprised by the sequences.
Peptides which are binding modules of the present invention also include
peptides having one or more substitutions, additions and/or deletions of
residues relative to the sequence of an exemplary peptide sequence as
disclosed herein, so long as the desired binding properties of the
binding module are retained. Thus, binding modules of the invention
include peptides that differ from the exemplary sequences disclosed
herein by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 amino acids, but that retain the ability of the
corresponding exemplary sequence to bind to a particular material or to
act as an analyte module. A binding module of the invention that differs
from an exemplary sequence disclosed herein will retain at least 25%,
50%, 75%, or 100% of the activity of a binding module comprising an
entire exemplary sequence disclosed herein as measured using an
appropriate assay.

[0048]That is, binding modules of the invention include peptides that
share sequence identity with the exemplary sequences disclosed herein of
at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence
identity. Sequence identity may be calculated manually or it may be
calculated using a computer implementation of a mathematical algorithm,
for example, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package of Genetics Computer Group, Version 10
(available from Accelrys, 9685 Scranton Road, San Diego, Calif., 92121,
USA). The scoring matrix used in Version 10 of the Wisconsin Genetics
Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc.
Nat'l. Acad. Sci. USA 89: 10915). Alignments using these programs can be
performed using the default parameters.

[0049]A peptide can be modified, for example, by terminal-NH2
acylation (e.g., acetylation, or thioglycolic acid amidation) or by
terminal-carboxylamidation (e.g., with ammonia or methylamine). Terminal
modifications are useful to reduce susceptibility by proteinase
digestion, and to therefore prolong a half-life of peptides in solutions,
particularly in biological fluids where proteases can be present.

[0050]Peptide cyclization is also a useful modification because of the
stable structures formed by cyclization and in view of the biological
activities observed for such cyclic peptides. Methods for cyclizing
peptides are described, for example, by Schneider & Eberle (1993)
Peptides. 1992: Proceedings of the Twenty-Second European Peptide
Symposium, Sep. 13-19, 1992, Interlaken, Switzerland, Escom, Leiden, The
Netherlands.

[0051]Optionally, a binding module peptide can comprise one or more amino
acids that have been modified to contain one or more halogens, such as
fluorine, bromine, or iodine, to facilitate linking to a linker molecule.
As used herein, the term "peptide" also encompasses a peptide wherein one
or more of the peptide bonds are replaced by pseudopeptide bonds
including but not limited to a carba bond (CH2--CH2), a depsi
bond (CO--O), a hydroxyethylene bond (CHOH--CH2), a ketomethylene
bond (CO--CH2), a methylene-oxy bond (CH2--O), a reduced bond
(CH2--NH), a thiomethylene bond (CH2--S), an N-modified bond
(--NRCO--), and a thiopeptide bond (CS--NH). See e.g.,
Garbay-Jaureguiberry et al. (1992) Int. J. Pept. Protein Res. 39:
523-527; Tung et al. (1992) Pept. Res. 5: 115-118; Urge et al. (1992)
Carbohydr. Res. 235: 83-93; Corringer et al. (1993) J. Med. Chem. 36:
166-172; Pavone et al. (1993) Int. J. Pept. Protein Res. 41: 15-20.

[0052]Representative peptides that specifically bind to surfaces of
interest (including titanium, stainless steel, collagen, and poly
glycolic acid (PGA)) and therefore are suitable for use as binding
modules in IFBMs of the invention are set forth in the sequence listing
and are further described herein below. While exemplary peptide sequences
are disclosed herein, one of skill will appreciate that the binding
properties conferred by those sequences may be attributable to only some
of the amino acids comprised by the sequences. Thus, a sequence which
comprises only a portion of an exemplary sequence disclosed herein may
have substantially the same binding properties as the full-length
exemplary sequence. Thus, also useful as binding modules are sequences
that comprise only 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acids
in a particular exemplary sequence, and such amino acids may be
contiguous or non-contiguous in the exemplary sequence. Such amino acids
may be concentrated at the amino-terminal end of the exemplary peptide
(for example, 4 amino acids may be concentrated in the first 5, 6, 7, 8,
9, 10, 11, or 12 amino acids of the peptide) or they may be dispersed
throughout the exemplary peptide but nevertheless be responsible for the
binding properties of the peptide. For example, a peptide that
specifically binds to BMP-2 may comprise all or part of a sequence motif
such as that described in Example 3 and set forth in SEQ ID NO:27 or 28.
Thus, a peptide that specifically binds to BMP-2 may have a sequence that
conforms to each requirement of the sequence motif as set forth in SEQ ID
NO:27 or 28, or it may have a sequence that conforms to 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 of the requirements of the sequence motif. The sequence
motif set forth in SEQ ID NO:27 can be described as having four
"requirements" which limit the amino acids that are present at positions
1, 4, 6, and 7. A peptide that specifically binds to BMP-2 may have a
sequence as set forth in SEQ ID NO:11 which conforms to all four of those
requirements, or it may have a sequence as set forth in SEQ ID NO:21
which conforms to three of those four requirements. Both of these types
of sequences are provided by the present invention.

[0053]In some embodiments, the IFBM has been constructed so as to mimic
the biological effects of protein growth factors. In these embodiments,
the analyte module comprises a peptide which comprises an amino acid
sequence which binds to the BMP receptor BMPRI and also comprises an
amino acid sequence which binds to the BMP receptor BMPRII (see, for
example, Example 6). These receptors are well-known in the art and are
also commercially available (for example, from R&D Systems, Minneapolis,
Minn., Cat. Nos. 315-BR and 811-BR). In these embodiments, the analyte
module has BMP activity as measured, for example, by techniques known in
the art and described in Example 6. While the invention is not bound by
any particular mechanism of operation, it is believed that by binding to
each of BMPRI and BMPRII, the analyte module will encourage the
heterodimerization of these receptors, thereby triggering signaling via
the BMP-SMAD pathway. In this manner, an IFBM could be constructed and
used to coat the surface of an implant so as to trigger signaling via the
BMP-SMAD pathway without the addition of BMP itself. Generally, in the
native BMP-SMAD pathway, heterodimerization of the BMP type I and type II
receptors is required for signaling (see, e.g., Chen et al. (2004) Growth
Factors 22: 233-241). Dimerization brings the cytoplasmic domains of the
type I and type II receptors into proximity, allowing the constitutively
active type II receptor kinase to phosphorylate the type I receptor. The
phosphorylation of the cytoplasmic domain of the type I receptor
activates its latent kinase activity which in turn activates Smad
proteins. After release from the receptor, the phosphorylated Smad
proteins associate with Smad4 and this complex is translocated into the
nucleus to function with other proteins as transcription factors and
regulate responsive genes (Chen et al. (2004) Growth Factors 22:
233-241). Collectively, this can be referred to as the downstream Smad or
BMP-SMAD signal transduction pathway and genes activated thereby.
Proteins produced as a result of activation of the Smad or BMP-SMAD
pathway can be referred to as Smad-activated downstream protein products.

[0054]Binding modules of the present invention that are peptides can be
synthesized by any of the techniques that are known to those skilled in
the art of peptide synthesis. Representative techniques can be found, for
example, in Stewart & Young (1969) Solid Phase Peptide Synthesis,
(Freeman, San Francisco, Calif.); Merrifield (1969) Adv. Enzymol. Relat.
Areas Mol. Biol. 32: 221-296; Fields & Noble (1990) Int. J. Pept. Protein
Res. 35: 161-214; and Bodanszky (1993) Principles of Peptide Synthesis,
2nd Rev. Ed. (Springer-Verlag, Berlin). Representative solid phase
synthesis techniques can be found in Andersson et al. (2000) Biopolymers
55: 227-250, references cited therein, and in U.S. Pat. Nos. 6,015,561;
6,015,881; 6,031,071; and 4,244,946. Peptide synthesis in solution is
described in Schroder & Lake (1965) The Peptides (Academic Press, New
York, N.Y.). Appropriate protective groups useful for peptide synthesis
are described in the above texts and in McOmie (1973) Protective Groups
in Organic Chemistry (Plenum Press, London). Peptides, including peptides
comprising non-genetically encoded amino acids, can also be produced in a
cell-free translation system, such as the system described by Shimizu et
al. (2001) Nat Biotechnol 19: 751-755. In addition, peptides having a
specified amino acid sequence can be purchased from commercial sources
(e.g., Biopeptide Co., LLC of San Diego, Calif.), and PeptidoGenics of
Livermore, Calif.).

[0055]The binding modules are connected by at least one linker to form an
IFBM of the invention. In some embodiments, IFBMs consisting of binding
modules which are peptides are synthesized as a single continuous
peptide; in these embodiments, the linker is simply one of the bonds in
the peptide. In other embodiments of the invention, a linker can comprise
a polymer, including a synthetic polymer or a natural polymer.
Representative synthetic polymers which are useful as linkers include but
are not limited to: polyethers (e.g., polyethylene glycol; PEG),
polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA),
polyamides (e.g., nylon), polyamines, polyacrylic acids, polyurethanes,
polystyrenes, and other synthetic polymers having a molecular weight of
about 200 daltons to about 1000 kilodaltons. Representative natural
polymers which are useful as linkers include but are not limited to:
hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin,
albumin, collagen, and other natural polymers having a molecular weight
of about 200 daltons to about 20,000 kilodaltons. Polymeric linkers can
comprise a diblock polymer, a multi-block copolymer, a comb polymer, a
star polymer, a dendritic polymer, a hybrid linear-dendritic polymer, or
a random copolymer.

[0056]A linker can also comprise a mercapto(amido)carboxylic acid, an
acrylamidocarboxylic acid, an acrlyamido-amidotriethylene glycolic acid,
and derivatives thereof. See, for example, U.S. Pat. No. 6,280,760. Where
a linker comprises a peptide, the peptide can include sequences known to
have particular biological functions, such as YGD and GSR.

[0058]The surfaces of medical devices are coated by any suitable method,
for example, by dipping, spraying, or brushing the IFBM onto the device.
The coating may be stabilized, for example, by air drying or by
lyophilization. However, these treatments are not exclusive, and other
coating and stabilization methods may be employed. Suitable methods are
known in the art. See, e.g., Harris et al. (2004) Biomaterials 25:
4135-4148 and U.S. patent application Ser. No. 10/644,703, filed Aug. 19,
2003 and published on May 6, 2004 with Publication No. 20040087505.

[0059]All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the art to
which this invention pertains. All publications and patent applications
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

[0060]Many modifications and other embodiments of the inventions set forth
herein will come to mind to one skilled in the art to which these
inventions pertain having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it is to
be understood that the inventions are not to be limited to the specific
embodiments disclosed and that modifications and other embodiments are
intended to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.

EXPERIMENTAL

Example 1

Isolation of Peptides that Bind Titanium

[0061]Ten different phage display libraries were screened for binding to
titanium beads. Titanium (Ti6Al4V) beads of approximately 5/32
of an inch diameter were washed with 70% ethanol, 40% nitric acid,
distilled water, 70% ethanol, and acetone to remove any surface
contaminants. One titanium bead was placed per well of 96-well
polypropylene plate (Nunc).

[0062]Nonspecific binding sites on the titanium and the surface of the
polypropylene were blocked with 1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS; Sigma Chemical Co., St. Louis, Mo., Cat.
#P-3813). The plate was incubated for 1 hour at room temperature with
shaking at 50 rpm. The wells were then washed 5 times with 300 μl of
PBS. Each library was diluted in PBS+1% BSA and was added at a
concentration of 1010 pfu/ml in a total volume of 250 μl. After a
3-hour incubation at room temperature and shaking at 50 rpm, unbound
phage were removed by washing 3 time with 300 μl of Phosphate Buffered
Saline-Tween® 20 (PBS-T; Sigma Chemical Co., St. Louis, Mo., Cat.
#P-3563). To recover the phage bound to the titanium beads, bound phage
were released by treating with 50 mM glycine, pH 2 for 10 minutes
followed by a 10 minute treatment with 100 mM ethanolamine, pH 12. The
eluted phage were pooled, neutralized with 200 μl of 200 mM NaPO4
pH 7. The eluted phage and the beads were added directly to E. coli
DH5αF' cells in 2×YT media. The mixture was incubated
overnight in a 37° C. shaker at 210 rpm. Phage supernatant was
then harvested after spinning at 8500×g for 10 minutes. Second and
third rounds of selection were performed in a similar manner to that of
the first round, using the 50 μl of amplified phage from the previous
round as input diluted with 200 μl of PBS+1% BSA. The fourth round of
selection was carried out in a similar fashion; however, the washes were
modified. After a 4 hour binding reaction, the beads were washed five
times with PBS-T (Sigma Chemical Co., St. Louis, Mo., Cat. #P-3563), the
beads were moved to a clean polypropylene plate with 2 ml wells, 1 ml of
PBS+1% BSA was added to each well and the washing was incubated overnight
at room temperature with shaking at 50 rpm. The next morning the phage
were eluted and amplified in the same manner described for rounds 1-3.
Individual clonal phage were then isolated and tested by plating out
dilutions of phage pools to obtain single plaques.

[0063]To detect phage that specifically bound to titanium, conventional
ELISAs were performed using an anti-M13 phage antibody conjugated to HRP,
followed by the addition of chromogenic agent ABTS. Relative binding
strengths of the phage were determined by testing serial dilutions of the
phage for binding to titanium in an ELISA.

[0064]The DNA sequence encoding peptides that specifically bound titanium
was determined. The sequence encoding the peptide insert was located in
the phage genome and translated to yield the corresponding amino acid
sequence displayed on the phage surface.

[0065]Representative peptides that specifically bind titanium are listed
in Table 1 and are set forth as SEQ ID NOs:1-8. The binding of phage
displaying these peptides to titanium beads is shown in FIG. 1.

[0066]The displayed peptides were then synthesized with a C-terminal
biotin residue and tested for binding to titanium. Results are shown in
FIG. 2. Briefly, peptide stock solutions were made by dissolving the
powder in 100% DMSO to make a 1 mM solution of peptide. Serial dilutions
of the peptide were made in PBS-T. Titanium beads blocked with 1% non-fat
dry milk in PBS were incubated with various concentrations of peptide for
1 hour at room temperature with shaking. The beads were washed 3 times
with PBS-T. Streptavidin-alkaline phosphatase (SA-AP) from USB (United
States Biochemical, catalog #11687) was added (1:1000 in PBS-T) and
incubated 1 hour at room temperature with shaking. The beads were washed
3 times with PBS-T and the amount of peptide:SA-AP was determined by
adding PNPP (Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891) and
allowing the color to develop for about 10 minutes. Quantitation was
carried out by transferring the solution to a clear microtiter plate and
reading the absorbance at 405 nm on a Molecular Dynamics Plate Reader.
The peptide "9003" is known in the art. This peptide was identified by
phage display as binding to the enzyme hexokinase; it serves as a
negative control for this experiment (see, e.g., Hyde-DeRuyscher et al.
(2000) Chem. Biol. 7: 17-25).

Example 2

Role of Cysteine Residues in Titanium-Binding Peptide 6007

[0067]To explore the role of the cysteine residues and disulfide formation
in the binding of peptide 6007 to titanium, a peptide was synthesized
AFF6010 (Table 2) in which the cysteine residues present in the
titanium-binding peptide AFF6007 were changed to serine residues. The
sequence of peptide AFF6010 (SSSDKSHKHWYSYESKYGGSGSSGK) is set forth in
SEQ ID NO:9, while the sequence of peptide AFF6007
(SSSDKCHKHWYCYESKYGGSGSSGK) is set forth in SEQ ID NO:10. The peptides
AFF6007 and AFF6010 were then conjugated to biotin and compared for
binding to titanium beads as follows.

[0068]Titanium beads were blocked with 1% BSA in PBS for 30 minutes at
room temperature. Stock solutions of peptide AFF6007 and AFF6010 were
prepared by dissolving 1-2 mg peptide in water. The final concentration
of each peptide was determined using the optical density at 280 nm and
the extinction coefficient of each peptide. AFF6007 and AFF6010 were
prepared at 200 μM. A dilution series was then prepared for each
peptide sample. Each peptide underwent a threefold dilution in 1% BSA in
PBS.

[0069]The peptides were incubated with the titanium beads for 1 hour at
room temperature. Beads were then washed two times with PBS/Tween® 20.
Streptavidin-alkaline phosphatase was then added to the beads at 1:500
for 30 minutes at room temperature. Beads were washed two times with
PBS/Tween® 20. PNPP was used to develop the assay and the absorbance
was recorded at 405 nm.

[0070]The results, which are shown in FIG. 3, demonstrate that peptides
AFF6007 and AFF6010 both bind to titanium. An estimate of the relative
affinity of a peptide for titanium can be made by determining the
concentration of peptide that gives one-half the maximal signal (Table
2). The complete elimination of the cysteine residues in AFF6007
decreases the affinity of the peptide for titanium by about 10-fold but
does not eliminate it (Table 2). Therefore, the cysteine residues are not
required for binding to titanium but do increase the affinity of the
peptide for titanium.

[0071]Ten different phage display libraries were screened for binding to
BMP-2. BMP-2 (Medtronic) was biotinylated with NHS-biotin (Pierce) to
produce a labeled protein with an average of one biotin per protein
molecule. This protein was immobilized on streptavidin (SA) coated plates
and used as target for phage display. As an alternative method to display
the protein, BMP-2 was also linked to sepharose beads using
NHS-succinimide chemistry according to the instructions of the
manufacturer (Amersham-Pharmacia, Ref. No. 18-1022-29, entitled "Coupling
through the Primary Amine of a Ligand to NHS-activated Sepharose 4 Fast
Flow," pp. 105-108) and the beads were used as a solid phase to separate
free from unbound phage. After 3 rounds of selection, individual clones
from each format were tested for binding to BMP-2 on SA coated plates
utilizing a conventional ELISA using an anti-M13 phage antibody
conjugated to HRP, followed by the addition of chromogenic agent ABTS.

[0072]The DNA sequence encoding peptides that specifically bound to BMP-2
was determined. The sequence encoding the peptide insert was located in
the phage genome and translated to yield the corresponding amino acid
sequence displayed on the phage surface. Representative peptides that
specifically bind BMP-2 are listed in Table 3 and are set forth as SEQ ID
NOs:11-26. In some embodiments, an exemplary binding module of the
invention comprises only that portion of the sequence shown in uppercase
letters.

[0073]The peptides that were identified fall into 2 different "sequence
clusters". Each sequence cluster contains a common sequence motif. For
the first sequence cluster of BMP-binding peptides, the common motif
(designated "Motif 1" and set forth in SEQ ID NO:27) is
Aromatic-X-X-Phe-X-"Small"-Leu (Aromatic=Trp, Phe, or Tyr; X=any amino
acid; "Small"=Ser, Thr, Ala, or Gly). Motif 1 is at least partially found
in SEQ ID NOs:11-24 as shown in Table 3 above. The second sequence
cluster motif (also set forth in SEQ ID NO:28) comprises the sequence
(Leu or Val)-X-Phe-Pro-Leu-(Lys or Arg)-Gly. This motif, designated Motif
2, is found in SEQ ID NOs:25 and 26 as shown in Table 3 above. Exemplary
binding modules also comprise sequences which meet the requirements of
this or other sequence motifs identified herein (i.e., which contain a
sequence which falls within these motifs).

[0074]Additional experiments were conducted to determine additional
characteristics of sequences that bind to BMP-2. Specifically, in order
to determine whether there were additional preferred amino acids
surrounding these motifs, further screening was conducted. Focused
libraries were designed and cloned into the mAEK phage display vector and
the resultant phage were screened for binding to BMP-2, as further
discussed below. The focused library for Motif 1 was designed to express
peptides containing the following sequence:
X-X-X-X-X-(W/L/C/Y/F/S)-X-X-(W/L/C/Y/F/S)-X-(A/G/N/S/T)-(L/F/I/V)-X-X-X-X-
-X, where X represents any of the 20 naturally occurring amino acids and
positions in parentheses are restricted to the amino acids listed within
the parentheses. These peptides were encoded by oligonucleotides
comprising the sequence
5'-GATCCTCGAGNNNKNNKNNKNNKNNKTNBNNKNNKTNBNNKRSYNTKNNKN
NKNNKNNKNNKTCTAGAGCGCTACG 3' (where "N" is any of the 4 nucleotides A, G,
C, or T; "K" is G or T; "R" is A or G; "S" is C or G; "B" is C, G, or T;
and "Y" is C or T). The focused library for Motif 2 was designed to
express peptides containing the following sequence:
X-X-X-(L/F/I/V)-X-(W/L/C/Y/F/S)-(P/S/T/A)-(L/F/I/M/V)-(I/M/T/N/K/S/R)-X-X-
-X-X-X-X-X-X. These peptides were encoded by oligonucleotides comprising
the sequence 5'-GATCCTCGANNNNKNNKNNKNTKNNKTNBNCKNTKANKNNKNNKNNKNNKNN
KNNKNNKNNKTCTAGAGCGCTACG 3'.

[0075]The following is provided as an exemplary library construction
scheme for the Motif 1 focused library. As will be appreciated by one of
skill in the art, a similar strategy can be used for other libraries. To
produce the focused library for Motif 1, an oligonucleotide comprising
the sequence above flanked by appropriate restriction enzyme sites was
synthesized. This oligonucleotide contained the sequence
5'-GATCCTCGAGNNNKNNKNNKNNKNNKTNBNNKNNKTNBNNKRSYNTKNNKNNKN
NKNNKNNKTCTAGAGCGCTACG-3'. In this sequence, the underlined sequences
CTCGAG and TCTAGA represent the XhoI and XbaI restriction enzyme sites
used to clone the library into the phage vector. A short primer is
annealed to the oligonucleotide and the complementary strand synthesized
using a DNA polymerase. The resulting double-stranded DNA molecule is
digested with XhoI and XbaI and cloned into the phage display vector. The
ligated DNA is transformed into an appropriate bacterial host and
amplified to generate the phage library.

[0076]The focused libraries for Motif 1 and Motif 2 were screened for
binding to BMP-2 using biotinylated BMP-2 immobilized on
streptavidin-coated plates as described above. After two rounds of
selection on BMP-2, the libraries had been enriched for phage displaying
peptides that bind to BMP-2. The pools of enriched phage were plated onto
a lawn of bacterial cells to isolate individual phage. Individual phage
clones were tested for binding to BMP-2 using an ELISA-type assay and an
anti-M13 phage antibody conjugated to HRP (Amersham Biosciences
#27-9421-01), followed by addition of the chromogenic reagent ABTS (Sigma
Chemical Co., St. Louis, Mo., Cat. #A3219).

[0077]The DNA sequence encoding peptides that specifically bound to BMP-2
was determined. The sequence encoding the peptide insert was located in
the phage genome and translated to yield the corresponding amino acid
sequence displayed on the phage surface.

[0078]Representative peptides from the motif-based focused libraries that
specifically bind BMP-2 are listed in Tables 4 and 5 and are set forth as
SEQ ID NOs:44-71 and 77-92. In some embodiments, an exemplary binding
module of the invention comprises only that portion of the sequence shown
in uppercase letters, or comprises only a sequence falling within a motif
or a consensus sequence identified based on these sequences (i.e.,
comprises a sequence falling within the scope of Motif 1, Motif 1a, or
Motif 2, or comprises the consensus sequence identified in SEQ ID NO:72,
74, or 93).

[0079]The results of an analysis of all the peptide sequences from Tables
3 and 4 that bind BMP-2 and contain Motif 1 was generated and is shown in
FIG. 7. From an alignment of the 40 BMP-binding sequences that contain
Motif 1, a consensus sequence can be derived
(Gly-Gly-Gly-Ala-Trp-Glu-Ala-Phe-Ser-Ser-Leu-Ser-Gly-Ser-Arg-Val; SEQ ID
NO: 72) that represents the predominant amino acid found at each position
after all the peptides are aligned. Among the 40 sequences, the most
conserved amino acids form a core binding motif which represents a subset
of all sequences containing Motif 1. This motif, designated "Motif 1a,"
has the sequence Trp-X-X-Phe-X-X-Leu (SEQ ID NO: 73). While the invention
is not bound by any particular mechanism of action, it is believed that
in this motif, the Trp, Phe, and Leu residues on the peptide participate
in specific interactions with the BMP-2 protein that are responsible for
the binding of the peptide to BMP. On this basis, it was hypothesized
that other peptides that contain this core binding motif will also bind
to BMP.

[0080]To test this idea, an oligonucleotide cassette was designed to
express a peptide which contained this core binding Motif 1a in the
context of a peptide sequence which also contained consensus residues
identified for other positions in the sequence that flanked the core
binding motif (see FIG. 8; SEQ ID NO: 74). Incidentally, none of the
BMP-binding peptides previously isolated by phage display actually
contain this exact sequence (see, e.g., Table 4). This oligonucleotide
cassette was cloned into the mAEK phage display vector and the resulting
phage, designated AP02-61, was tested for binding to BMP-2 and compared
to other phage displaying BMP-binding peptides (results for some phage
are shown in FIG. 9). At least one phage tested (designated AP02-37)
showed binding at a level equivalent to or below that of the display
vector mAEK. In some embodiments, an exemplary binding module of the
invention comprises only that portion of the sequence shown in uppercase
letters.

[0081]From an alignment of the peptides from Tables 3 and 5 that contain
Motif 2, a consensus sequence can be derived
(Gly-Gly-Ala-Leu-Gly-Phe-Pro-Leu-Lys-Gly-Glu-Val-Val-Glu-Gly-Trp-Ala; SEQ
ID NO: 93; see FIG. 10) that represents the predominant amino acid found
at each position after all the peptides are aligned. Among the sequences
examined, the most conserved amino acids form a core binding motif
designated "Motif 2a," which has the sequence Leu-X-Phe-Pro-Leu-Lys-Gly
(SEQ ID NO: 94).

[0082]Motif 2 appears to be more restricted in sequence than Motif 1 in
that Motif 2 imposes requirements on six positions whereas Motif 1 only
imposes requirements on three positions. The Pro and Gly residues in
Motif 2 appear to be required for binding since every Motif 2-containing
BMP-binding peptide contains the Pro and Gly residues found in the core
binding motif. Using the consensus sequence information for Motif 2,
BMP-binding peptides can be designed by incorporating the Motif 2 core
binding motif into the peptide sequence.

Production of Synthetic Peptides and BMP-2 Binding Assays

[0083]A representative set of the displayed peptides were then synthesized
with a C-terminal biotin residue and tested for binding to BMP-2. Results
are shown in FIG. 4. Briefly, peptide stock solutions were made by
dissolving the powder in 100% DMSO to make a 10 mM solution of peptide,
water was then added for a final stock concentration of peptide of 1 mM
in 10% DMSO. Serial dilutions of the peptide were made in PBS-T. A
dilution series of BMP-2 with concentrations ranging from 100 nM to 0.1
nM was immobilized onto the wells of microtiter plates (Immulon-4®
HBX from Dynex Technologies, Chantilly, Va.) and blocked with 1% BSA.
These plates were incubated with various concentrations of peptide for 1
hour at room temperature with shaking. The beads were washed 3 times with
PBS-T. Streptavidin-alkaline phosphatase (SA-AP) from USB (United States
Biochemical, catalog #11687) was added (1:1000 in PBS-T) and incubated 1
hour at room temperature with shaking. The plates were washed 3 times
with PBS-T and the amount of peptide:SA-AP was determined by adding PNPP
(Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891) and allowing the
color to develop for about 10 minutes. Quantitation was carried out by
reading the absorbance at 405 nm on a Molecular Dynamics Plate Reader.
The results are summarized in FIG. 4.

[0084]To confirm these BMP binding results, the peptides were also tested
in an alternate assay format in which the peptide and BMP2 were allowed
to bind in solution and then assayed. Briefly, the peptides were
synthesized with a biotin group attached to the ε amino group of
a lysine residue at the C-terminus of the peptide. The biotinylated
peptides (0-12 pmoles) were mixed with BMP-2 (0-25 pmoles) in solution
and allowed to incubate at 37° C. for 30 minutes in a
polypropylene plate. The solutions were transferred to a
streptavidin-coated plate and incubated for 1 hour at 37° C. to
capture the biotinylated peptides. Plates were washed in TBS-Tween® 20
and then incubated with an anti-BMP antibody (1:1000 dilution; R&D
systems) for 1 hour at RT. After washing, an alkaline phosphatase-labeled
secondary antibody was then added to the plate and incubated at RT for 30
minutes. The plates were washed with TBS-Tween® 20 and the antibody
binding was detected using the chromogenic AP substrate pNPP.
Representative results are shown in FIG. 11. From this data, the affinity
of each BMP-binding peptide for BMP-2 was calculated (Table 6).

[0085]Bone Morphogenetic Proteins (BMPs) are members of the TGF-beta
superfamily which includes BMPs, Transforming Growth Factor-beta
(TGF-β) and Growth/Differentiation factors (GDFs). The proteins in
the TGF-β superfamily are very similar structurally. The folded
structure of the protein backbone is almost identical among all the
members of the family. Based on the similarity in structure between the
BMPs, we tested the ability of some of the BMP-2-binding peptides to bind
BMP-4 and BMP-7. Biotinylated peptides 2007 and 2011 were tested for
binding to BMP-2, BMP-4, and BMP-7 as described above. Both 2007 and 2011
bound to all three BMPs while a peptide that binds to an unrelated target
(AFF-9001) did not bind to any of the BMPs (FIG. 12).

Example 4

Generation of an IFBM that Immobilizes BMP-2 onto Collagen

[0086]To design a molecule with collagen and BMP-2 binding properties, an
IFBM was created that comprised a peptide that binds to collagen and a
peptide that binds to BMP-2. Examples of this "hybrid peptide" IFBM are
shown in Table 7.

[0087]As shown in Table 7, each IFBM contains the collagen binding domain
from AFF0016 followed by a short linker sequence which is then linked to
a BMP binding sequence from the above example in a "hybrid peptide."
These molecules were synthesized in both orientations to assess the
effect of N- or C-terminal locations on the ability of the IFBM to bind
to collagen or BMP-2.

[0088]To determine if these IFBM's increased the amount of BMP retained by
a collagen sponge, we mixed the IFBM with BMP, added the mixture to a
sponge, allowed them to bind for 1.5 hours, washed the sponge and
detected the bound BMP with anti-BMP antibodies. Briefly, stock IFBM
solutions were prepared by weighing 1-2 mg peptide and solubilizing in
water. The final peptide concentration was determined by analyzing the
peptide absorbance at 280 nm and the extinction coefficient. For each
row, 20 μL of peptide were added to each well of a polypropylene
microtiter plate. BMP was then added to each of these wells in a
threefold dilution series, starting with 32 μM BMP. The IFBM and BMP
were allowed to mix at room temperature for 30 minutes.

[0089]To each well, a 2/16'' diameter collagen sponge (Medtronic) was
added. The collagen and peptide solutions were allowed to incubate for
1.5 hours at room temperature. Sponges were then rinsed three times with
200 μL Medtronic buffer at 2200 rpm for 1 minute. To each sponge, a
primary antibody directed at BMP (diluted 1:1000; R&D Systems #MAB3552)
was added for 1 hour at room temperature. A secondary antibody conjugated
to alkaline phosphatase (1:5000) was then incubated in the system for 0.5
hour at room temperature. PNPP was used to develop the system and
absorbances were read at 405 nm. Results are shown in FIG. 5.

[0091]BMP on the sponge than the sponges without IFBM. IFBM AFF7008 and
AFF7017 increase the amount of BMP on the sponge when compared to no
IFBM, but to a lesser extent than AFF7010. The increased retention of BMP
to the sponge is not seen by adding AFF2006, a BMP-binding peptide that
does not contain a collagen-binding sequence.

[0092]To show that this effect is dose dependent not only on the amount of
BMP put onto the sponge but also on the amount of IFBM present, a series
of two-dimensional dose response curves was obtained in which the
concentrations of both the IFBM and BMP were varied. These results are
shown in the FIG. 6A-6D and demonstrate that the binding of BMP to the
collagen sponge is dependent on both BMP concentration and IFBM
concentration. Increasing the concentration of the IFBM (AFF7005,
AFF7006, AFF7009, or AFF7010) leads to a larger amount of BMP-2 that is
retained on the collagen.

Example 5

Peptides that Bind to Stainless Steel

[0093]Selection of stainless steel-binding peptides was performed as
described above for the titanium-binding peptides except that 5/32 inch
stainless steel beads were used instead of titanium beads. The stainless
steel binding peptides that were isolated are shown in Table 8. In some
embodiments, an exemplary binding module of the invention comprises only
that portion of the sequence shown in uppercase letters.

[0094]Selection of Teflon (GoreTex®; polytetrafluorethylene
(PTFE))-binding peptides was performed as described above for the
titanium-binding peptides except that sections of GoreTex fabric were
used instead of titanium beads. The Teflon-binding peptides that were
isolated are shown in Table 9. In some embodiments, an exemplary binding
module of the invention comprises only that portion of the sequence shown
in uppercase letters.

[0095]Identification of peptides that bind to BMPRI and/or BMPRII: In
order to identify peptides that specifically bind to Bone Morphogenic
Protein Receptor I (BMPRIA) and/or Bone Morphogenic Protein Receptor II
("BMPRII"), phage display libraries are screened to identify phage
encoding peptides that bind to the extracellular domains of each
receptor. The extracellular domains of these receptors are known in the
art (Rosenweig et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 7632-7636;
Ten Dijke et al. (1994) J. Biol. Chem. 269: 16985-16988). Various phage
libraries are screened. Where appropriate, a phage library can be
selected that is designed around a specific amino acid motif or that was
made with a particular amino acid bias. BMPRIA and BMPRII (R&D Systems,
Cat. Nos. 315-BR/CF and 811-BR) are dissolved in carbonate coating buffer
(100 mM NaHCO3, pH 9.6); 100 μl of this solution is added to the
wells of a 96-well Immulon®-4 microtiter plate (Dynex Technologies,
Chantilly, Va.). The plate is incubated overnight at 4° C. and
then the nonspecific binding sites on the surface of the polystyrene are
blocked with 1% Bovine Serum Albumin (BSA) in carbonate coating buffer.
The plate is then incubated for an hour at room temperature with shaking
at 50 rpm. The wells are then washed 5 times with 300 μl of PBS-T
(Sigma Chemical Co., St. Louis, Mo., Cat. #P-3563). Each library is
diluted in PBS-T and added at a concentration of 1010 pfu/ml in a total
volume of 100 ul. The plates are then incubated at room temperature with
shaking at 50 rpm for 3 hours; unbound phage is then removed with 5
washes of PBS-T. Bound phage are recovered by denaturation with 0.1 M
glycine buffer pH 2.2 (see Phage Display of Peptides and Proteins: A
Laboratory Manual, 1996, eds. Kay et al. (Academic Press, San Diego,
Calif.)). Eluted phage are neutralized with phosphate buffer and then
added to E. coli DH5a cells in 2×YT media. This mixture is
incubated overnight at 37° C. in a shaker at 210 rpm. Phage
supernatant is harvested by centrifuging at 8500×g for 10 minutes.
Second and third rounds of selection are performed similarly to the first
round of selection using the phage from the previous round of selection
as the input phage. Phage display techniques are well known in the art,
for example, as described in Sparks et al. (1996) "Screening
phage-displayed random peptide libraries," pp. 227-253 in Phage Display
of Peptides and Proteins: A Laboratory Manual, eds. Kay et al. (Academic
Press, San Diego, Calif.).

[0096]To identify phage that specifically bind to BMPRIA or BMPRII,
conventional ELISAs are performed using an anti-M13 phage antibody
conjugated to horseradish peroxidase (HRP), followed by the addition of
chromogenic agent ABTS (Sigma Chemical Co., St. Louis, Mo., Cat. #A3219).
Relative binding strengths of the phage are determined by testing serial
dilutions of the phage for binding to BMP receptors in an ELISA. The DNA
encoding each selected peptide is isolated and sequenced to determine the
amino acid sequence of the selected peptide.

[0097]These peptides are then linked together to create an analyte module
that will bind to each of BMPRI and BMPRII, forming a heterodimer of
these two receptors so as to induce signaling. Candidate peptides are
synthesized and biotinylated and their binding to the BMP receptors
confirmed. Briefly, the biotinylated peptides are synthesized with a
linker between the BMP receptor binding sequence and the attached biotin
moiety. This linker has the amino acid sequence GSSGK, which serves to
separate the biotin moiety from the receptor binding portion of the
peptide and which is flexible. Peptides are synthesized using solid-phase
peptide synthetic techniques on a Rainin Symphony Peptide Synthesizer
(Rainin Instrument Co., Emeryville, Calif.) using standard Fmoc
chemistry. N-α-Fmoc-amino acids (with orthogonal side chain
protecting groups) can be purchased from Novabiochem
(Calbiochem-Novabiochem, Laufelfingen, Switzerland). After all residues
are coupled, simultaneous cleavage and side chain deprotection will be
achieved by treatment of a trifluoroacetic acid (TFA) cocktail. Crude
peptide is precipitated with cold diethyl ether and purified by
high-performance liquid chromatography on a Shimadzu
Analytical/Semi-preparative HPLC unit on a Vydac C18 silica column
(preparative 10 μm, 250 mm×22 mm; Grace Vydac Co., Hesperia,
Calif.) using a linear gradient of water/acetonitrile containing 0.1%
TFA. Homogeneity of the synthetic peptides is evaluated by analytical
RP-HPLC (Vydac C18 silica column, 10 μm, 250 mm×4.6 mm) and the
identity of the peptides is confirmed with MALDI-TOF-MS, for example, as
performed commercially at the UNC-CH Proteomics Core Facility.

[0098]Generation of peptides that bind to BMPRI and/or BMPRII with high
affinity: Peptides that are initially identified as binding to BMPRI
and/or BMPRII may have low binding affinities, e.g., in the mid- to
low-μM range, whereas it may be preferable that peptides for use in an
IFBM have higher binding affinities, e.g., in the nM range. To identify
such peptides, libraries of variants of the initially identified peptides
are constructed and screened by affinity selection against BMPRI and/or
BMPRII.

[0099]Determination of binding affinity is evaluated using procedures
known in the art. For example, BMPRI, BMPRII, and appropriate control
proteins are dissolved in carbonate coating buffer (100 mM NaHCO3,
pH 9.6) and added to the wells of a 96-well polypropylene plate. After
incubation overnight at 4° C., the wells are blocked with 1% BSA
in PBS-T. Each receptor and control is tested for binding over a range of
peptide concentrations from 0 to 200 μM in sterile PBS (pH 7.2). The
wells are then washed to remove unbound peptide and a
streptavidin-alkaline phosphatase conjugate solution (SA-AP) from USB
(United States Biochemical #11687) is added to each well to quantify the
amount of bound peptide. Streptavidin-alkaline phosphatase activity is
measured using the chromogenic reagent p-nitrophenyl phosphate reagent
(Sigma-Aldrich, Inc., SigmaFast tablets, catalog #N1891) and measuring
absorbance at 405 nm. To determine a binding curve and rough KD,
absorbance is plotted as a function of the concentration for each
peptide. The impact of other factors on binding can be assessed, such as
for example, pH, temperature, salt concentration, buffer components, and
incubation time.

[0100]To create and identify peptides that bind to BMPRI and/or BMPRII
with higher affinity, phage libraries are created based on an amino acid
motif identified among the initial peptides isolated as binding to BMPRI
and/or BMPRII and screened further for peptides with improved binding
properties. Such techniques are known in the art (see, for example,
Hyde-DeRuyscher et al. (2000) Chem. Biol. 7: 17-25; Dalby et al. (2000)
Protein Sci. 9: 2366-2376).

[0101]Characterization of agonist activity of hybrid peptides comprising
BMPRI-binding peptides and BMPRII-binding peptides: Synthetic peptides
are chemically synthesized that comprise both a BMPRI-binding peptide and
a BMPRII-binding peptide connected with a flexible linker (e.g., a linker
having the sequence GSSGSSG). Alternatively, the two receptor-binding
peptides may be linked through the α and ε amino groups of
a lysine (e.g., as in Cwirla et al. (1997) Science 276: 1696-1699 or in
Wrighton et al. (1997) Nat. Biotechnol. 15: 1261-1265). These peptides
are about 40 amino acids in length and are readily synthesized and
purified.

[0102]These peptides are then assayed for BMP activity such as, for
example, the induction of alkaline phosphatase activity in mouse
mesenchymal C3H10T1/2 cells as known in the art and described, for
example, by Cheng et al. (2003) J. Bone Joint Surg. Am. 85-A: 1544-1552
and Ruppert et al. (1996) Eur. J. Biochem. 237: 295-302. Briefly,
C3H10T1/2 cells are added to a 96-well plate (3×104 cells per
well in a volume of 2000 in Gibco® MEM/EBSS medium (Invitrogen Corp.,
Carlsbad, Calif., Cat #11095-080) with 10% FBS and appropriate
antibiotics and antimycotics. Cells are permitted to adhere to the plate
for at least 3 hours by incubating at 37° C. in a 5% CO2
atmosphere. Media is then aseptically aspirated and BMP-2 or peptides are
added at various concentrations in high-glucose Gibco® DMEM
(Invitrogen Corp., Carlsbad, Calif., Cat. #11965-092) plus 2% FBS. Cells
are incubated with the tested compounds for three days, at which time the
media is aspirated and the cells are washed three times with 300 μl of
PBS (Gibco® PBS, Cat. #14190-144, Invitrogen Corp., Carlsbad,
Calif.). 100 μl of pNPP (p-Nitrophenyl Phosphate Sigma Fast Tablet Set
Cat #N-1891) in H2O is added to each well and the color is allowed
to develop for up to 18 hours at 37° C. before absorbance is read
at 405 nm.

[0103]EC50 values are then determined using methods known in the art.
Typical EC50 values for this assay for BMP-2 range between 1
μg/ml and 10 μg/ml (see, e.g., Wiemann et al. (2002) J. Biomed.
Mater. Res. 62: 119-127). It is known in the art that BMP-2 isolated from
different sources can show different levels of activity, and one of skill
in the art can adjust procedures accordingly to take these differences
into account to achieve the desired result. For example, it is known in
the art that recombinant human BMP-2 ("rhBMP-2") prepared using CHO cells
has activity which differs 5-10 fold from the activity of recombinant
human BMP-2 prepared using E. coli (see, e.g., Zhao and Chen (2002),
"Expression of rhBMP-2 in Escherichia coli and Its Activity in Inducing
Bone Formation," in Advances in Skeletal Reconstruction Using Bone
Morphogenic Proteins, ed. T. S. Lindholm).

[0104]Immobilization of hybrid peptides onto collagen: Hybrid peptides
that show BMP activity are synthetically linked to a peptide that binds
to collagen. Briefly, a peptide containing the collagen binding module
and the BMPRI-binding module is synthesized with an orthogonal protecting
group on an amino acid in the linker between the modules, such as
Fmoc-Lys(Dde)-OH. The Dde protecting group on the c amino group of the
lysine side chain can be selectively removed and a BMPRII-binding peptide
coupled to the c amino group. Alternatively, a linear peptide can be
synthesized that comprises the collagen-binding module, the BMPRI-binding
module, and the BMPRII-binding module.

[0105]The collagen-bound hybrid peptide is then tested for its BMP
activity, such as by assaying for the induction of alkaline phosphatase
activity in mouse mesenchymal C3H10T1/2 cells while the hybrid peptide is
bound to a collagen matrix. Briefly, 5-mm disks of collagen are washed
with PBS and added to the cell-based BMP activity assay.

Example 8

Sterilization of Surfaces Coated with IFBMs

[0106]IFBM-coated surfaces were treated with electron-beam sterilization
procedures and gamma sterilization procedures. The binding performance of
the coated surfaces was assessed before and after the sterilization
procedures. Assays were performed on polystyrene and titanium surfaces.
For the polystyrene assay, a binding module ("AFF-0002-PS") was
biotinylated and relative binding was assessed by exposing the binding
module to streptavidin-conjugated alkaline phosphatase. The results
showed that the amount of biotinylated peptide that was bound to the
polystyrene surface was essentially identical before and after the
sterilization procedures. Similar results were obtained for an assay of a
binding module ("AFF-0006-Ti") on titanium; in this assay, the
performance of the coated surface before sterilization was approximately
equal to its performance after sterilization.

Example 9

Preliminary Toxicity Testing

[0107]A PEGylated polystyrene-binding peptide was coated onto various
polystyrene surfaces and tested as follows for adverse effects including
cytotoxicity, hemolysis, and coagulation. The procedures were performed
in Albino Swiss Mice (Mus musculus). As further discussed below, none of
the IFBMs tested showed any signs of toxicity.

[0108]To assay for acute systemic toxicity, polystyrene squares (each
square 4×4 cm; a total of 60 cm2) were incubated for 70-74
hours at 37° C. in 20 mL of one of two vehicles: 0.9% USP normal
saline or cotton seed oil (National Formulary). Five mice were each
injected systemically with either vehicle or vehicle-extract at a dose
rate of 50 mL extract per kg body weight. Mice were observed for signs of
toxicity immediately after injection and at 4, 24, 48, and 72 hours
post-injection. None of the animals injected with the vehicle-extract
showed a greater biological reaction than those that received vehicle
alone.

[0109]Coated surfaces were assayed for partial thromboblastin time
according to ISO procedure 10993-4 (International Organization for
Standardization, Geneva, Switzerland). Briefly, fresh whole human blood
was drawn into vacutainer tubes containing sodium citrate and were spun
down to isolate plasma, which was stored on ice until use. Coated
polystyrene squares (as described above) were then incubated in the
plasma at a ratio of 4 cm2 per 1 mL for 15 minutes at 37° C.
in polypropylene tubes and agitated at 60 rpm. The plasma extract was
then separated, placed on ice, and tested on a Cascade® M-4 manual
hemostasis analyzer (Helena Laboratories, Beaumont, Tex.). Clotting time
was not significantly different than that observed for pure plasma or the
standard reference control.

[0110]Cytotoxicity was assayed in L-929 Mouse Fibroblast Cells as
specified in ISO 10993-5. Briefly, 60.8 cm2 of polystyrene-coated
squares was extracted into 20.3 mL of Eagle's Minimum Essential Medium+5%
FBS at 37° C. for 24 hours. Positive, negative and intermediate
cell-line test dishes were incubated at 37° C. in a humidified 5%
CO2 atmosphere. Cultures were evaluated for cytotoxic effects by
microscopic observation at 24, 48, and 72 hours. The positive control
showed a strong cytotoxic reaction score of "4" while test cells
maintained a healthy ("0" score) appearance across all time points (score
of "0"). Intermediate control cells scored as "2" across all time points.

[0111]Hemolysis testing measures the ability of a material or material
extract to cause red blood cells to rupture. The test performed was ASTM
F-756 Direct Contact Method. Saline was used to extract leachable
substances. Coated polystyrene surface was extracted and then added to
citrated rabbit blood (3.2%, diluted with PBS to obtain a total blood
hemoglobin concentration of 10 mg/ml). A score of 0.4% was observed which
falls into the passing category of 0-2%. The negative control returned a
score of 0.1% and the positive control returned a score of 12.2%.